The X6 "Thermostabilizing" Domains of Xylanases Are Carbohydrate-Binding Modules: Structure and Biochemistry of the Clostridium thermocellum X6b Domain
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- 作者：Simon J. Charnock ; David N. Bolam ; Johan P. Turkenburg ; Harry J. Gilbert ; Luis M. A. Ferreira ; Gideon J. Davies ; and Carlos M. G. A. Fontes
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：May 2, 2000
- 年：2000
- 卷：39
- 期：17
- 页码：5013 - 5021
- 全文大小：485K
- 年卷期：v.39,no.17(May 2, 2000)
- ISSN：1520-4995
文摘
Many polysaccharide-degrading enzymes display a modular structure in which a catalytic moduleis attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknownfunction (termed "X6" modules) that had been implicated in thermostability. We have investigated theproperties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with thecatalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolyticactivity was determined. The data showed that X6b, but not X6a, increased the activity of the enzymeagainst insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast,X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealedthat the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibitedKd values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 
M forxylotriose; and 50 M for xylobiose. Negative 
H and S values indicate that the interaction of X6bwith xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure ofX6b has been solved by X-ray crystallography to a resolution of 2.1 Å. The protein is a 
-sandwich thatpresents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be thexylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposedthat this and related modules be re-assigned as family 22 carbohydrate-binding modules.
M forxylotriose; and 50 M for xylobiose. Negative H and S values indicate that the interaction of X6bwith xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure ofX6b has been solved by X-ray crystallography to a resolution of 2.1 Å. The protein is a -sandwich thatpresents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be thexylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposedthat this and related modules be re-assigned as family 22 carbohydrate-binding modules.